title: Isolation of human conjunctival mast cells and epithelial cells: tumor necrosis factor-alpha from mast cells affects intercellular adhesion molecule 1 expression on epithelial cells.
abstract: OBJECTIVE
  To isolate and purify mast cells and epithelial cells from human cadaveric donor conjunctival tissue and to characterize interactions between these cell types in vitro.
  
  
  METHODS
  Monodispersed cell suspensions obtained by enzymatic digestion of conjunctival tissue were applied to a single-density Percoll gradient. Epithelial cells obtained from the top layer of the gradient were cultured to confluence. Mast cells obtained from the pellet were equilibrated in culture medium and further purified using a two-step Percoll gradient. Using reverse transcription-polymerase chain reaction (RT-PCR), RNA from the purified mast cell preparation was probed for tumor necrosis factor-alpha (TNF alpha) message. Fluorescence activated cell sorting (FACS) analysis of intracellular immunostained mast cells was used to detect the TNF alpha protein. An examination for intercellular adhesion molecule 1 (ICAM-1) on epithelial cells was performed after 24-hour incubations with either recombinant TNF alpha supernatants from calcium ionophore A23187 (CaI)-stimulated mast cells or appropriate controls using FACS analysis.
  
  
  RESULTS
  Highly purified human conjunctival mast cells and epithelial cells (each > 95%) were obtained from human cadaveric donor tissue. RT-PCR analysis of purified mast cell RNA revealed the expression of TNF alpha mRNA. An evaluation of mast cells for intracellular protein demonstrated positive staining for tryptase and TNF alpha. ICAM-1 was found on purified epithelial cells, and incubation of epithelial cell monolayers with supernatants from Cal-stimulated mast cells resulted in upregulation of this receptor. This upregulation was blocked by incubation with TNF alpha-neutralizing antibody.
  
  
  CONCLUSIONS
  This work provides the methods for isolating and purifying mast cells and epithelial cells from human donor tissue and the opportunity for studying mechanisms of conjunctival inflammation by evaluating the interactions between these cells.
authors.affiliation: Department of Medicine, School of Medicine, University of Wisconsin-Madison 53792, USA.
authors.forename: E B
authors.initials: EB
authors.lastname: Cook
authors.forename: J L
authors.initials: JL
authors.lastname: Stahl
authors.forename: S T
authors.initials: ST
authors.lastname: Miller
authors.forename: J E
authors.initials: JE
authors.lastname: Gern
authors.forename: K A
authors.initials: KA
authors.lastname: Sukow
authors.forename: F M
authors.initials: FM
authors.lastname: Graziano
authors.forename: N P
authors.initials: NP
authors.lastname: Barney
mesh_terms: D000001:Calcimycin / Q000494:pharmacology; D002469:Cell Separation; D002478:Cells, Cultured; D053818:Chymases; D003228:Conjunctiva / Q000166:cytology*; D017931:DNA Primers / Q000737:chemistry; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D008407:Mast Cells / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism; D014158:Transcription, Genetic; D053802:Tryptases; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*; D015854:Up-Regulation
chemical_list: D017931:DNA Primers; D007476:Ionophores; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D000001:Calcimycin; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases